RESUMEN
Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.
Asunto(s)
Leucocitos , Hígado , Animales , Ratones , Hígado/inmunología , Leucocitos/inmunología , Hepatocitos/inmunología , Separación Celular/métodos , Análisis de la Célula Individual/métodos , Ratones Endogámicos C57BL , Masculino , Linfocitos/inmunología , Linfocitos/citologíaRESUMEN
Immune checkpoint inhibitors are effective first-line therapy for solid cancers. However, low response rate and acquired resistance over time has led to the need for additional therapeutic options. Here, we evaluated synergistic antitumor efficacy of EGFR × MET targeting bispecific antibody, amivantamab with PD-L1 immunotherapy, pembrolizumab in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma tumor-bearing humanized patient-derived xenograft (PDX) models. We demonstrated that pembrolizumab or amivantamab alone was ineffective and that combination treatment induced a significant reduction of tumor growth in both models (P < 0.0001 and P < 0.01, respectively). It appeared that combination of amivantamab and pembrolizumab significantly enhanced infiltration of granzyme B-producing CD8 T cells was in the TME of HNSCC PDX (P < 0.01) and enhanced neoantigen-associated central memory CD8 T cells in circulating immune cells. Analysis of single-cell RNA transcriptomics suggested that the tumor cells dramatically upregulated EGFR and MET in response to PD-L1 immunotherapy, potentially creating a metabolic state fit for tumor persistence in the tumor microenvironment (TME) and rendered pembrolizumab ineffective. We demonstrated that EGFRHIGHMETHIGH subcluster displayed an increased expression of genes implicated in production of lactate [SLC16A3 and lactate dehydrogenase A (LDHA)] compared to the EGFRLOWMETLOW cluster. Accumulation of lactate in the TME has been associated with immunosuppression by hindering the infiltration of tumor killing CD8 T and NK cells. This study proved that amivantamab reduced glycolytic markers in the EGFRHIGHMETHIGH subcluster including SLC16A3 and LDHA and highlighted remodeling of the TME by combination treatment, providing rationale for additional therapy of amivantamab with PD-1 immunotherapy. SIGNIFICANCE: Amivantamab in synergy with pembrolizumab effectively eradicated EGFRHIGHMETHIGH tumor subcluster in the tumor microenvironment of head and neck squamous cell carcinoma and overcame resistance against anti-PD-1 immunotherapy.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Neoplasias Pulmonares , Carcinoma de Células Escamosas de Cabeza y Cuello , Microambiente Tumoral , Humanos , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Ratones , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Antígeno B7-H1/metabolismo , Línea Celular TumoralRESUMEN
Progress in the generation of Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding this process. HSPCs emerge within the embryonic vasculature through an Endothelial-to-Hematopoietic Transition (EHT). The transcriptional regulator Tal1 exerts essential functions in the earliest stages of blood development, but is considered dispensable for the EHT. Nevertheless, Tal1 is expressed with its binding partner Lmo2 and it homologous Lyl1 in endothelial and transitioning cells at the time of EHT. Here, we investigated the function of these genes using a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development. We showed for the first time that the expression of TAL1 in endothelial cells is crucial to ensure the efficiency of the EHT process and a sustained hematopoietic output. Our findings uncover an important function of Tal1 during the EHT, thus filling the current gap in the knowledge of the role of this master gene throughout the whole process of hematopoietic development.
Asunto(s)
Células Endoteliales , Hematopoyesis , Animales , Diferenciación Celular/genética , Células Endoteliales/metabolismo , Endotelio , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Ratones , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismoRESUMEN
HOX proteins are homeodomain transcription factors critically involved in patterning animal embryos and controlling organogenesis. While the functions of HOX proteins and the processes under their control begin to be well documented, the modalities of HOX protein activity regulation remain poorly understood. Here we show that HOXA2 interacts with PPP1CB, a catalytic subunit of the Ser/Thr PP1 phosphatase complex. This interaction co-localizes in the cytoplasm with a previously described HOXA2 interactor, KPC2, which belongs to the KPC E3 ubiquitin ligase complex. We provide evidence that HOXA2, PPP1CB and KPC2 define a molecularly and functionally interacting complex. Collectively, our experiments support that PPP1CB and KPC2 together inhibit the activity of HOXA2 by activating its nuclear export, but favored HOXA2 de-ubiquitination and stabilization thereby establishing a store of HOXA2 in the cytoplasm.
Asunto(s)
Citoplasma/genética , Proteínas de Homeodominio/genética , Proteína Fosfatasa 1/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Citosol/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Procesamiento Proteico-Postraduccional/genética , Estabilidad ProteicaRESUMEN
In vitro differentiation of mouse embryonic stem cells (mESCs) towards blood cells constitutes a well-established system to study the endothelial-to-hematopoietic transition (EHT) at the onset of blood development. Assessing the emergence of small non-adherent round blood cells in the culture without disturbing it is essential to evaluate the progression of EHT and also to test conditions potentially enhancing or repressing this process. Here, we describe how to quantify the formation of mouse hematopoietic progenitors during EHT in normal conditions or following over-expression of eight essential transcription factors using time-lapse microscopy and image analysis.
RESUMEN
Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Análisis de la Célula Individual/métodos , Factores de Transcripción/genética , Animales , Análisis por Conglomerados , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Endotelio/citología , Endotelio/embriología , Endotelio/metabolismo , Redes Reguladoras de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Proto-Oncogénica c-fli-1/genéticaRESUMEN
The homeodomain transcription factor Hoxa2 interacts with the RING-finger type E3 ubiquitin ligase RCHY1 and induces its proteasomal degradation. In this work, we dissected this non-transcriptional activity of Hoxa2 at the molecular level. The Hoxa2-mediated decay of RCHY1 involves both the 19S and 20S proteasome complexes. It relies on both the Hoxa2 homeodomain and C-terminal moiety although no single deletion in the Hoxa2 sequence could disrupt the RCHY1 interaction. That the Hoxa2 homeodomain alone could mediate RCHY1 binding is consistent with the shared ability all the Hox proteins we tested to interact with RCHY1. Nonetheless, the ability to induce RCHY1 degradation although critically relying on the homeodomain is not common to all Hox proteins. This identifies the homeodomain as necessary but not sufficient for what appears to be an almost generic Hox protein activity. Finally we provide evidence that the Hoxa2-induced degradation of RCHY1 is evolutionarily conserved among vertebrates. These data therefore support the hypothesis that the molecular and functional interaction between Hox proteins and RCHY1 is an ancestral Hox property.
Asunto(s)
Proteínas de Homeodominio/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Núcleo Celular/enzimología , Desarrollo Embrionario , Estabilidad de Enzimas , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Semivida , Proteínas de Homeodominio/química , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ProteolisisRESUMEN
Regulation of transcription factor activity relies on molecular interactions or enzymatic modifications which influence their interaction with DNA cis-regulatory sequences, their transcriptional activation or repression, and stability or intracellular distribution of these proteins. Regarding the well-conserved Hox protein family, a restricted number of activity regulators have been highlighted thus far. In the framework of a proteome-wide screening aiming at identifying proteins interacting with Hoxa2, KPC2, an adapter protein constitutive of the KPC ubiquitin-ligase complex, was identified. In this work, KPC2 was confirmed as being a genuine interactor of Hoxa2 by co-precipitation and bimolecular fluorescence complementation assays. At functional level, KPC2 diminishes the transcriptional activity and induces the nuclear exit of Hoxa2. Gene expression analyses revealed that Kpc2 is active in restricted areas of the developing mouse embryo which overlap with the Hoxa2 expression domain. Together, our data support that KPC2 regulates Hoxa2 by promoting its relocation to the cytoplasm.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas de Homeodominio/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Activación Transcripcional/genética , Animales , Citoplasma/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Unión ProteicaRESUMEN
Understanding the mode of action of Hox proteins requires the identification of molecular and cellular pathways they take part in. This includes to characterize the networks of protein-protein interactions involving Hox proteins. In this chapter we propose a strategy and methods to map Hox interaction networks, from yeast two-hybrid and high-throughput yeast two-hybrid interaction screening to bioinformatic analyses based on the software platform Cytoscape.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Mapeo de Interacción de Proteínas/métodos , Biología Computacional/métodos , Bases de Datos de Proteínas , Ensayos Analíticos de Alto Rendimiento , Unión Proteica , Mapas de Interacción de Proteínas , Reproducibilidad de los Resultados , Técnicas del Sistema de Dos Híbridos , Levaduras/metabolismoRESUMEN
Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Humanos , Inmunohistoquímica , Unión Proteica , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Hox proteins are transcription factors involved in crucial processes during animal development. Their mode of action remains scantily documented. While other families of transcription factors, like Smad or Stat, are known cell signaling transducers, such a function has never been squarely addressed for Hox proteins. RESULTS: To investigate the mode of action of mammalian Hoxa1, we characterized its interactome by a systematic yeast two-hybrid screening against ~12,200 ORF-derived polypeptides. Fifty nine interactors were identified of which 45 could be confirmed by affinity co-purification in animal cell lines. Many Hoxa1 interactors are proteins involved in cell-signaling transduction, cell adhesion and vesicular trafficking. Forty-one interactions were detectable in live cells by Bimolecular Fluorescence Complementation which revealed distinctive intracellular patterns for these interactions consistent with the selective recruitment of Hoxa1 by subgroups of partner proteins at vesicular, cytoplasmic or nuclear compartments. CONCLUSIONS: The characterization of the Hoxa1 interactome presented here suggests unexplored roles for Hox proteins in cell-to-cell communication and cell physiology.